1. Technical Field
This invention relates to a novel method for identifying or screening an agonist for and/or antagonist to peroxisome proliferator activated receptor (PPAR).
2. Background Art
Peroxisome, an organelle found in the cells of animals and plants, contains a group of enzymes participating in the lipometabolism and absorption of lipids such as cholesterol. An increase in peroxisome is also induced by diet or physiological factors. It is known that a group of chemicals diversified in structure including antilipemic (fibrates), insecticides and plasticizers such as phthalic acids when they are administered dramatically increase the size and number of peroxisome in liver and kidney and at the same time elevate the ability of metabolizing fatty acids in peroxisome through intermediary of an increase in the expression of enzymes necessary for the β-oxidation cycle. Hence, they are called peroxisome proliferator. Among studies on the mechanism of such a peroxisome proliferation, a nuclear receptor that is activated by the group of chemicals has been identified and named peroxisome proliferator activated receptor (PPAR).
From its structure, etc., PPAR is considered to be a member of nuclear receptor (nuclear hormone receptor) super family. Like other nuclear receptors, it is activated by its binding to a ligand, and its binding to a response sequence (PPRE: peroxisome proliferator response element) existing upstream of a target gene domain activates transcription of the target gene. PPAR is known to form a heterodimer with a retinoid X receptor (RXR) and binds to PPRE in the form of the heterodimer. Also, like other nuclear receptors, PPAR is considered to have the interaction with a group of transcription coactivators (coactivators) in order to exhibit its transcription activation activity.
Hitherto, three kinds of PPAR subtypes called PPARα, PPARδ (or NUC-1, PPARβ, FAAR) and PPARδ have been identified and their genes (cDNA) have been cloned (Lemberger et al., Annu. Rev. Cell. Dev. Biol., vol. 12, pp. 335–363, 1996). Of the three kinds, PPARδ is expressed particularly in an adipose tissue and considered to be a factor that closely participates in differentiation of adipocytes (Tontonoz et al., Genes and Development, vol. 8, pp. 1224–1234, 1994; Tontonoz et al., Cell, vol. 79, pp. 1147–1156, 1994).
On the other hand, various thiazolidinedione derivatives show hypoglycemic effect in a model animal of non-insulin-dependent diabetes mellitus (NIDDM) and are expected as a NIDDM remedy having an insulin resistance releasing effect. These thiazolidinedione derivatives act as ligands to PPARγ and specifically activate PPARγ, which has been discovered in recent studies (Lehmann et al., Journal of Biological Chemistry, vol. 270, pp. 12953–12956, 1995). Since a strong correlation is observed between such a PPARγ activation ability of thiazolidinedione derivatives and the hypoglycemic effect in a hereditary obese mouse, PPARγ is considered to be a target molecule of the pharmaceutical effect of the thiazolidinedione derivatives (Willson et al., Journal of Medicinal Chemistry, vol. 39, pp. 665–668, 1996). This also relates to the fact that an adipose tissue where PPARγ is specifically expressed is an organ that plays an important role in maintaining energy balance. From these findings, a compound specifically acting as an agonist for PPARγ is considered to be very useful as a remedy for diabetes mellitus.
However, to date, those methods known as screening methods for PPAR acting agents each involve the problems that operation is complicated and simultaneous treatment of multiple samples is difficult.
For example, there has been known a method for examining PPAR activation ability of a sample using animal cells having introduced therein reporter plasmid containing a reporter gene linked to a PPAR expression vector and a PPAR response element (PPRE), with using as an index a change in the amount of expression of a reporter gene in the cells (WO 96/22884, Tontonoz et al., Genes and Development, vol. 8, pp. 1224–1234, 1994). As its improved method, there has been known a method using animal cells having introduced therein vector for expressing fused protein in which the DNA binding domain of GAL4, i.e., the transcription factor of yeast, and the ligand binding domain of PPAR linked together, along with having introduced a reporter plasmid containing a reporter gene linked to the response element of GAL4 (GAL4 binding element) (WO 96/33724, Lehmann et al., Journal of Biological Chemistry, vol. 270, pp. 12953–12956, 1995; Willson et al., Journal of Medicinal Chemistry, vol. 39, pp. 665–668, 1996). In these methods, an extrinsic gene is introduced into animal cells. Upon the introduction of gene, it is sometimes the case that the integration of a gene into a chromosome has taken place, the gene is influenced by the site where the gene is integrated. Therefore, it is necessary to use a transformed cell in which gene is not influenced by the chromosome. To acquire such a transformed animal cell and express an extrinsic gene stably are accompanied by technical difficulties. Since coactivators, RXR, etc. derived from host animal are considered to participate in the activation of transcription in these methods, there is the possibility that the action of the test substance to PPAR alone cannot be detected surely.
As a method for directly detecting the binding between PPAR and a ligand without using any animal cell or reporter gene, there has been known a method for examining binding and antagonism between a fused protein comprising the ligand binding domain of PPAR and glutathione-S-transferase (GST) and a test compound labeled with a radioisotope (Willson et al., Journal of Medicinal Chemistry, vol. 39, pp. 665–668, 1996; Buckle et al., Bioorganic & Medical Chemistry Letters, vol. 6, pp. 2121–2126, 1996). Recently, it has been elucidated that like other nuclear receptor RXR, etc., PPAR interacts with SRC-1, one of coactivators, ligand-dependently. Based on this finding, Krey et al. reported a method for detecting the action of a test compound as a ligand using a fused protein comprising the ligand binding domain of PPAR and glutathione-S-transferase (GST) and SRC-1 labeled with a radioisotope (Krey et al., Molecular Endocrinology, Vol. 11, pp. 779–791, 1997). However, these methods each use a label of radioisotope and therefore it is accompanied by a danger and has a limitation in treating power since preparation of a labeled compound or coactivator on a large scale is difficult.
As described above, upon screening PPAR acting agents, a screening method which is simple, high precision, and efficient has been desired.
An object of this invention is to provide a novel method for identifying and screening an agonist and/or antagonist to peroxisome proliferator activated receptor (PPAR).
The present inventors have uniquely found that in addition to SRC-1, one of the coactivators, that is already known to interact with PPAR, CBP (CREB-binding protein) interacts with PPAR ligand-dependently and identified the binding domain of the coactivator to PPAR. Further, based on these findings, they have completed a method for identifying or screening a novel PPAR acting agent that detects a ligand-dependent interaction between PPAR and a coactivator using a Two-hybrid system of yeast.